We are interested in exploring the interactions between the immune system of the human malaria vector Anopheles gambiae and Plasmodium parasites to establish how they affect vector competence. Some major areas of interest include:[unreadable] [unreadable] Reactive oxygen species (ROS) and immunity in An. gambiae:[unreadable] We found that ROS modulate An. gambiae immunity, and that the mosquito response to P. berghei involves a local reduction of detoxification of hydrogen peroxide by catalase in the midgut that contributes to limit Plasmodium infection. The manuscript describing this work has been published in the Journal of Biological Chemistry.[unreadable] [unreadable] Interaction of Plasmodium with mosquito midgut epithelial cells:[unreadable] Blood feeding triggers the expression of an immuno-modulatory peroxidase, (IMPer) in the midgut on A. gambiae. This enzyme is secreted into the midgut lumen and prevents the activation of antibacterial immune responses in midgut epithelial cells, allowing the proliferation of the bacterial flora. In antibiotic-treated mosquitoes, IMPer silencing dramatically reduces P. berghei and P. falciparum infection and induces high levels of nitric oxide synthase (NOS) expression. Furthermore, double silencing of IMPer and NOS rescues parasite survival to control levels. This physiological induction of IMPer following a blood meal provides a permissive environment for Plasmodium development, as it prevents activation of both antibacterial and NOS-mediated anti-malarial responses. The manuscript describing this work is in preparation.[unreadable] [unreadable] We identified a serine protease (SP) that is highly induced in the midgut in response to ookinete invasion. SP silencing drastically reduces both P. berghei and P. falciparum infection. Silencing of the serine protease Inhibitor 6 (SRPN6) has the opposite effect as SP silencing, enhancing infection. Furthermore, SP silencing abolishes the induction of SRPN6 mRNA in response to Plasmodium invasion, suggesting that both genes are part of the same signaling cascade, with SP being upstream of SRPN6. We are currently investigating how SP silencing affects the cell biology of parasite midgut invasion. SP expression is also highly induced in the salivary gland in response to P. falciparum sporozoite invasion.[unreadable] [unreadable] Effect of pre-exposure of An. gambiae to Plasmodium in on subsequent infections: [unreadable] Two groups of mosquitoes were fed on the same mouse infected with P. berghei. One group was kept at a permissive temperature that allows ookinete development and midgut invasion. The second group was placed at a non-permissive temperature immediately after blood-feeding. The group in which midgut invasion took place has a significantly lower infection when challenged with a second Plasmodium infection either 1 or 2 weeks later. The reduction in oocyst numbers is independent of the number of oocysts from the first challenge and is not due to differences in the size of the blood meal ingested. Administration of antibiotics before the first or the second challenge abolishes this difference in infectivity. We conclude that bacteria must be present at the time of the first exposure to establish a long-lasting enhancement in the anti-parasitic response and that they are also required at the time of the second exposure, to elicit this response. We observed similar phenomena in mosquitoes infected with P. falciparum. The effects on Plasmodium appear to be indirect and mediated by responses to bacteria.[unreadable] [unreadable] STAT signaling pathways and immune responses to Plasmodium: [unreadable] In the mosquito An. gambiae, the ancestral STAT gene (AgSTAT-A) duplicated through a retrotransposition event. In adult females, the new intronless STAT gene (AgSTAT-B) regulates expression of AgSTAT-A. AgSTAT-A, in turn, mediates the transcriptional activation of NOS and SOCS in response to bacterial and plasmodial infection, indicating that this is an ancient signaling cascade. Activation of the STAT-A pathway, however, is not essential for mosquitoes to survive a bacterial challenge. AgSTAT-A silencing reduces early midgut infection with Plasmodium, but paradoxically enhances the overall infection by drastically increasing oocyst survival. Silencing of SOCS, a suppressor of STAT, has the opposite effect, reducing Plasmodium infection. The STAT pathway defines a novel late phase in the An. gambiae immune responses to Plasmodium. A manuscript describing this work has been submitted.[unreadable] [unreadable] Identification of mosquito genes required for Plasmodium oocyst development: [unreadable] Dr. David Schneider group performed a forward genetic screen, using Drosophila as a surrogate mosquito, to identify host factors required for the growth of the avian malaria parasite, Plasmodium gallinaceum. They identified 18 presumed loss-of-function mutants that reduced the growth of the parasite in flies. We evaluated the effect of silencing 5 Anopheles gambiae homologs by dsRNA injection on P. berghei infection. Loss-of-function of four of these genes in the mosquito affected Plasmodium growth, suggesting that Drosophila can be used effectively as a surrogate mosquito to identify relevant host factors in the mosquito. The manuscript describing this work is in press in Genetics. [unreadable] [unreadable] We compared the effect of silencing 7 genes in An. gambiae on P. berghei and P. falciparum infection. We find that 3 genes had a similar effect on both parasite species, but 4 of them have a different phenotype. When orthologues of these genes were silenced in An. stephensi, their effect on P. yoelii infection was very similar to what was observed in the An. gambiae-P.falciparum combination. These two vector-parasite combinations represent highly compatible systems, and this could explain their similar response. In contrast, An. gambiae is partially refractory to P. yoelii infection, and most parasites are either lysed or melanized. Silencing of the immune mediator LRIM1 prevents parasite killing, indicating that the refractoriness of An. gambiae to P. yoelii infection is mediated by the mosquitos immune response to the parasite. The manuscript describing this work is in preparation.[unreadable] [unreadable] One of the genes identified in the Drosophila screen is a member of the tetraspanin family of proteins. Tetraspanins are proteins with four transmembrane domains that are associated extensively with one another and with other membrane proteins to form specific microdomains distinct from lipid rafts. They are involved in diverse processes from cell adhesion to signal transduction. We identified three tetraspanin genes (Tet1, Tet, 2 and Tet3) that are induced in the midgut in response to P. falciparum infection. Silencing of Tet1 enhances P. berghei infection, Tet2 silencing reduces infection, while Tet3 silencing has no effect. Tet1 silencing also enhances P. falciparum infection (we are currently testing Tet2 and Tet3). We have generated polyclonal antibodies to Tet1 and will explore its distribution in the membrane in both healthy and Plasmodium-invaded cells.[unreadable] [unreadable] Identiffication of P. falciparum gene(s) that allow African strains to escape melanization in the An. gambiae refactory strain.[unreadable] A strain of An. gamibiae (L35 strain) was selected in 1986 to be highly refractory (R) to P. cynomolgy infection. This strain also melanizes several Plasmodium species, including P. falciparum strains from the New World. Interestingly, parasite strains of African origin are poorly melanized. We phenotype the parental parasites from a 7G8 X GB4 genetic cross that was Dr. Wellems laboratory. In the R strain, the Brazilian 7G8 parasites are melanized, while those of the GB4 strain from Ghana survive. We are currently phenotyping the progeny of the cross to identify the QTL(s) responsible for the phenotypic difference between these two P. falciparum strains.